Peptide, apoEp 1.B, compositions and uses thereof

ABSTRACT

A peptide derived from apolipoprotein E termed apoEp1.B which includes amino acids 239-252 of the apolipoprotein E is described. The apoEp1.B peptide is a potent immune modulator that acts on a variety of immune cells. Interestingly, apoEp1.B is a dual modulator, capable of both inducing and suppressing an immune response. In particular, apoEp1.B has been shown to induce differentiation of stem cells into dendritic cells, to induce tumor cell differentiation and activation, to inhibit inflammation and to inhibit autoimmune disease.

This application claim the benefit of provisional application Ser. No.60/069,531 filed Dec. 12, 1997.

FIELD OF THE INVENTION

The invention relates to methods and compositions for immune modulation.

BACKGROUND OF THE INVENTION

The immune system is a complex, multifactorial defense system thatprotects the body from a wide range of infectious diseases includingviruses, bacteria, parasites and fungi. Although critical for oursurvival, in certain instances, such as autoimmune disease, transplantrejection, allergies and inflammation, the immune system can be thecause of illness. In such instances it is desirable to suppress ortolerize the immune response.

The immune system is comprised of a large variety of cells derived fromundifferentiated hematopoietic stem cells and includes phagocytes (suchas neutrophil polymorphs, monocytes, macrophages and dendritic cells)and lymphocytes such as T cells and B cells and natural killer cells.

Dendritic cells are interesting immune cells as, depending on thecircumstances, they can either activate or suppress an immune response.With regard to immune activation, dendritic cells (DCs) are potentlymphocyte stimulators and are extremely effective antigen presentingcells. Recently, considerable interest has been generated in thepotential use of dendritic cells for the therapy of cancer andinfectious diseases. DCs pulsed with tumour peptides elicit protectiveand antitumour immunity in mice (Mayordama et al., 1995). Patients withB cell lymphomas have been successfully vaccinated with autologousantigen-pulsed DC directly isolated from the blood (Hsu et al., 1996).Flt3 ligand, which induces DC maturation, resulted in tumour regressionand antitumour immune response in mice (Lynch et al., 1997).Unfortunately, advances in treatment of tumors with DCs have beenlimited by their trace level in vivo. Efforts in this area are directedat increasing DC numbers and level of activation. With regard totolerance, dendritic cells have recently been shown to be involved inthe induction of central as well as peripheral tolerance and may beuseful in treating autoimmunity, allergies and transplantationrejection.

SUMMARY OF THE INVENTION

The present inventors have prepared a peptide derived fromapolipoprotein E termed apoEp1.B which includes amino acids 239-252 ofthe apolipoprotein E. The inventors have found that surprisingly,apoEp1.B is a potent immune modulator that acts on a variety of immunecells. Interestingly, apoEp1.B is a dual modulator, capable of bothinducing and suppressing an immune response. In particular, apoEp1.B hasbeen shown to induce differentiation of immature cells into dendriticcells, to induce tumor cell differentiation and activation, to inhibitinflammation and to inhibit autoimmune disease.

Accordingly, the present invention provides an isolated apoEp1.B peptidecomprising amino acids 239-252 of an apolipoprotein E protein. In apreferred embodiment, the present invention provides an isolatedapoEp1.B peptide having the amino acid sequence TQQIRLQAEIFQAR(SEQ.ID.NO.: 1) (murine) or AQQIRLQAEAFQAR (SEQ.ID.NO.: 2) (human). Theinvention also includes analogs, fragments, elongations and derivativesof a peptide of the invention. Analogs and derivatives of the peptidesinclude peptides having the following sequences: TAQIRLQAEIFQAR(SEQ.ID.NO.: 3); TQAIRLQAEIFQAR (SEQ.ID.NO.: 4); TQQARLQAEIFQAR(SEQ.ID.NO.: 5) and TQQIALQAEIFQAR (SEQ.ID.NO.: 6). Fragments andelongations of the peptides include peptides that have the followingsequences: QTQQIRLQAEIFQAR (SEQ.ID.NO.: 7) and QQIRLQAEIFQAR(SEQ.ID.NO.: 8). The present invention also provides a nucleic acidmolecule encoding the apoEp1.B peptide, or an analog, fragment orderivative thereof.

The present invention further provides a method of immune modulationcomprising administering an effective amount of an apoEp1.B peptide or anucleic acid encoding an apoEp1.B peptide to a cell or animal in needthereof.

According to one embodiment, the peptide can induce immune tolerance. Inparticular, the present inventors have demonstrated that the apoEp1.Bpeptide can activate monocytes to differentiate into tolerogenicdendritic cells. The induction of tolerogenic dendritic cells can have awide variety of therapeutic applications including inflammation,autoimmune disease and transplantation.

In another embodiment,the apoEp1.B peptide is useful in inhibitinginflammation. In a preferred embodiment, the peptide can inhibitatherosclerotic plaque formation in vivo.

In a further embodiment, the apoEp1.B peptide can be used to prevent ortreat an autoimmune reaction or disease. The present inventors have alsodemonstrated that the apoEp1.B peptide can protect NOD mice fromdeveloping diabetes. In a preferred embodiment, the autoimmune diseaseis diabetes.

In a further aspect, the apoEp1.B peptide can be used to induce animmune response by activating immune cells. In one embodiment, thepeptide, in combination with other cytokines such as IL-4, GM-CSF, TNFαand Flt3 ligand may induce immature dendritic cells to differentiateinto mature immunogenic dendritic cells. Mature dendritic cells can beused in a wide variety of applications including tumor immunotherapy.

In another aspect, the apoEp1.B peptide can be used to treat tumors ofimmune origin by inducing their differentiation. In particular, thepresent inventors have demonstrated that the apoEp1.B peptide can inducethe differentiation and activation of monocytic, monoblastic leukemiaand lymphoma tumor cells.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIGS. 1A and B are photographs of spleen cells incubated with apoEp1.B(B) or a control peptide (A) for 48 hours.

FIGS. 1C and D are photographs of PU5-1.8 cells incubated with apoEp1.B(D) or a control peptide (C) for 48 hours.

FIGS. 2(A-D) is a FACS analysis of J77A cells incubated with apoEp1.B (Band D) or with a control peptide (A and C) for 48 hours.

FIG. 3 is a FACS analysis of PU5-1.8 cells induced with apoEp1.B orapoEp1.D and stained with various markers.

FIG. 4 shows FACS analysis of BAlb/c splenocytes incubated with 15apoEp1.B or apoEp1.D.

FIG. 5 shows a FACS analysis illustrating that apoEp1.B induced DCsurface molecule expression is dose-dependent.

FIG. 6 shows a FACS analysis which demonstrates that apoEp1.B is notspecies-specific.

FIG. 7 are bar graphs demonstrating chemokine production from PU5-1.8cells incubated with apoEp1.B, apoEp1 and a control.

FIG. 8A is a FACS analysis showing SCC and FSC profiles of apoEp1.D andapoEp1.B primed PEC cells.

FIG. 8B is a FACS analysis showing surface marker expression of apoEp1.D(unlabelled peak) and apoEp1.B (labelled peak) primed PEC cells.

FIG. 9 is a bar graph showing activation of spleen cells in the presenceof apoEp1.B and/or ConA.

FIG. 10 shows a section of iliofemoral arteries: (A) untreated, nosurgery;

(B) untreated, surgery and (C) treated with apoEp1.B and surgery(arterial section shown).

FIG. 11 shows histograms which illustrate the induction of Th2-likecells by apoEp1.B immunization.

FIG. 12 shows a histogram which illustrates allostimulatory abilities ofapoEp1.B treated cells.

FIG. 13A is a graph which illustrates apoEp1.B protection of NOD micefrom spontaneous diabetes.

FIG. 13B is a graph which illustrate apoEp1.D protection of NOD micefrom adoptively transferred diabetes.

FIG. 14 is a graph showing the percentage of mice protected fromdiabetes versus time, in the presence and absence of apoEp1.B.

FIGS. 15A-D illustrate the effect of different concentrations ofapoEp1.B on proliferation of U-937 cells.

FIG. 16 shows a FACS analysis which illustrates that single amino aciddeletions or elongations decrease the activity of apoEp1.B.

DETAILED DESCRIPTION OF THE INVENTION

As hereinbefore mentioned, the present inventors have prepared a peptidefrom both human and murine apolipoprotein E termed apoEp1.B (239-252)which is a potent immune modulator and can be used in a wide variety ofapplications.

I. PEPTIDES OF THE INVENTION

Broadly stated, the present invention provides an isolated apoEp1.Bpeptide comprising amino acids 239-252 of an apolipoprotein E protein oran analog, fragment, elongation or derivative thereof.

In one aspect, the present invention provides an isolated apoEp1.Bpeptide having the amino acid sequence TQQIRLQAEIFQAR (murine)(SEQ. ID.No. 1) or AQQIRLQAEAFQAR (human)(SEQ. ID. No. 2) or an analog, fragment,elongation or derivative of the peptide. The invention also includes anucleic acid molecule encoding the apoEp1.B peptide, or an analog,fragment, elongation or derivative thereof.

The term “analog” includes any peptide having an amino acid residuesequence substantially identical to the human or mouse apoEp1.B sequencespecifically shown herein in which one or more residues have beenconservatively substituted with a functionally similar residue and whichdisplays the ability to mimic apoEp1.B as described herein. Examples ofconservative substitutions include the substitution of one non-polar(hydrophobic) residue such as alanine, isoleucine, valine, leucine ormethionine for another, the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, between glycine and serine, the substitutionof one basic residue such as lysine, arginine or histidine for another,or the substitution of one acidic residue, such as aspartic acid orglutamic acid for another. The phrase “conservative substitution” alsoincludes the use of a chemically derivatized residue in place of anon-derivatized residue provided that such polypeptide displays therequisite activity.

Analogs of the peptides include peptides having the following sequences:TAQIRLQAEIFQAR (SEQ.ID.NO.:3); TQAIRLQAEIFQAR (SEQ.ID.NO.:4);TQQARLQAEIFQAR (SEQ.ID.NO.:5) and TQQIALQAEIFQAR (SEQ.ID.NO.:6).

“Derivative” refers to a peptide having one or more residues chemicallyderivatized by reaction of a functional side group. Such derivatizedmolecules include for example, those molecules in which free aminogroups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,chloroacetyl groups or formyl groups. Free carboxyl groups may bederivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included asderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexamples: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andomithine may be substituted for lysine. Polypeptides of the presentinvention also include any polypeptide having one or more additionsand/or deletions or residues relative to the sequence of a polypeptidewhose sequence is shown herein, so long as the requisite activity ismaintained.

The term “fragment” refers to any subject peptide having an amino acidresidue sequence shorter than that of a peptide whose amino acid residuesequence is shown herein.

The term “elongation” refers to any subject peptide having an amino acidsequence longer by one or two amino acids (either at the carboxy oramino terminal end) than that of a peptide of the present invention.Preferably, the elongation occurs at the amino terminal end.

Fragments and elongations of the peptides include peptides that have thefollowing sequences: QTQQIRLQAEIFQAR (SEQ.ID.NO.:7) and QQIRLQAEIFQAR(SEQ.ID.NO.:8).

The term “apoEp1.B peptide” or “peptide of the invention” as used hereinincludes a peptide comprising amino acid residues 239 to 252 of anapolipoprotein E protein and includes all analogs, fragments,elongations or derivatives of the apoEp1.B peptide including thesequences provided above in SEQ.ID.NOS.:1-8. Preferably, the apoEp1.B isthe murine apoEp1.B sequence TQQIRLQAEIFQAR (SEQ. ID. No. 1) or thehuman apoEp1.B sequence AQQIRLQAEAFQAR (SEQ. ID. No. 2).

The apoEp1.B peptide may be modified to make it more therapeuticallyeffective or suitable. For example, it may be cyclized as cyclizationallows a peptide to assume a more favourable conformation. Cyclizationof the apoEp1.B peptide may be achieved using techniques known in theart. In particular, disulphide bonds may be formed between twoappropriately spaced components having free sulfhydryl groups. The bondsmay be formed between side chains of amino acids, non-amino acidcomponents or a combination of the two. In addition, the apoEp1.Bpeptide of the present invention may be converted into pharmaceuticalsalts by reacting with inorganic acids including hydrochloric acid,sulphuric acid, hydrobromic acid, phosphoric acid, etc., or organicacids including formic acid, acetic acid, propionic acid, glycolic acid,lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid,tartaric acid, citric acid, benzoic acid, salicylic acid,benzenesulphonic acid, and tolunesulphonic acids.

The apoEp1.B proteins of the invention may also be prepared byconventional techniques. For example, the peptides may be synthesized bychemical synthesis using techniques well known in the chemistry ofproteins such as solid or solution phase synthesis (see for example J.M. Stewart, and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed.,Pierce Chemical Co., Rockford Ill. (1984) and G. Barany and R. B.Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Grossand J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254 forsolid phase synthesis techniques; and M Bodansky, Principles of PeptideSynthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer,Eds., The Peptides: Analysis, Synthesis, Biology, supra, Vol 1, forclassical solution synthesis and Merrifield, 1964, J. Am. Chem. Assoc.85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987,Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 1 and 11, Thieme,Stuttgart).

The apoEp1.B peptides of the invention may also be produced byrecombinant DNA technology. To prepare the peptides of the invention byrecombinant DNA techniques, a DNA sequence encoding the apoEp1.B peptidemust be prepared. Consequently, the present invention also providespurified, and isolated nucleic acid having a nucleotide sequenceencoding an apoEp1.B peptide comprising an amino acid sequenceTQQIRLQAEIFQAR (SEQ. ID. No. 1) or an amino acid sequence AQQIRLQAEAFQAR(SEQ. ID. No. 2).

The present invention also provides an expression vector comprising aDNA molecule encoding an apoEp1.B peptide adapted for transfection ortransformation of a host cell.

Accordingly, the nucleic acid molecules of the present invention may beincorporated in a known manner into an appropriate expression vectorwhich ensures expression of the protein. Possible expression vectorsinclude but are not limited to cosmids, plasmids, or modified viruses(e.g. replication defective retroviruses, adenoviruses andadeno-associated viruses). The vector should be compatible with the hostcell used. The expression vectors are “suitable for transformation of ahost cell”, which means that the expression vectors contain a nucleicacid molecule of the invention and regulatory sequences selected on thebasis of the host cells to be used for expression, which is operativelylinked to the nucleic acid molecule. Operatively linked is intended tomean that the nucleic acid is linked to regulatory sequences in a mannerwhich allows expression of the nucleic acid.

The invention therefore contemplates a recombinant expression vector ofthe invention containing a nucleic acid molecule of the invention, or afragment thereof, and the necessary regulatory sequences for thetranscription and translation of the inserted protein-sequence.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes (Forexample, see the regulatory sequences described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Selection of appropriate regulatory sequences isdependent on the host cell chosen as discussed below, and may be readilyaccomplished by one of ordinary skill in the art. Examples of suchregulatory sequences include: a transcriptional promoter and enhancer orRNA polymerase binding sequence, a ribosomal binding sequence, includinga translation initiation signal. Additionally, depending on the hostcell chosen and the vector employed, other sequences, such as an originof replication, additional DNA restriction sites, enhancers, andsequences conferring inducibility of transcription may be incorporatedinto the expression vector.

The recombinant expression vectors of the invention may also contain aselectable marker gene which facilitates the selection of host cellstransformed or transfected with a recombinant molecule of the invention.Examples of selectable marker genes are genes encoding a protein such asG418 and hygromycin which confer resistance to certain drugs,β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase,or an immunoglobulin or portion thereof such as the Fc portion of animmunoglobulin preferably IgG.

Recombinant expression vectors can be introduced into host cells toproduce a transformant host cell. The term “transformant host cell” isintended to include prokaryotic and eukaryotic cells which have beentransformed or transfected with a recombinant expression vector of theinvention. The terms “transformed with”, “transfected with”,“transformation” and “transfection” are intended to encompassintroduction of nucleic acid (e.g. a vector) into a cell by one of manypossible techniques known in the art. Prokaryotic cells can betransformed with nucleic acid by, for example, electroporation orcalcium-chloride mediated transformation. Nucleic acid can be introducedinto mammalian cells via conventional techniques such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran mediatedtransfection, lipofectin, electroporation or microinjection. Suitablemethods for transforming and transfecting host cells can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition,Cold Spring Harbor Laboratory press (1989)), and other laboratorytextbooks.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells. For example, the proteins of the invention may be expressedin bacterial cells such as E. coli, insect cells (using baculovirus),yeast cells or mammalian cells. Yeast and fungi host cells suitable forcarrying out the present invention include, but are not limited toSaccharomyces cerevisae, the genera Pichia or Kluyveromyces and variousspecies of the genus Aspergillus. Mammalian cells suitable for carryingout the present invention include, among others: COS (e.g., ATCC No. CRL1650 or 1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa(e.g., ATCC No. CCL 2), 293 (ATCC No. 1573) and NS-1 cells. Othersuitable host cells can be found in Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1991).

Another aspect of the invention provides a nucleotide sequence whichhybridizes under high stringency conditions to a nucleic acid sequencewhich encodes an apoEp1.B peptide of the invention. Appropriatestringency conditions which promote DNA hybridization are known to thoseskilled in the art, or can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6. For example,6.0×sodium chloride/sodium citrate (SSC) at about 45° C., followed by awash of 2.0×SSC at 50° C. may be employed. The stringency may beselected based on the conditions used in the wash step. By way ofexample, the salt concentration in the wash step can be selected from ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be at high stringency conditions, at about 65° C.

II. UTILITY OF THE PEPTIDES A. Therapeutic Methods

The inventors have surprisingly found that the apoEp1.B peptide of theinvention is a potent immune modulator that acts on a variety of immunecells. Interestingly, apoEp1.B is a dual modulator, capable of bothinducing and suppressing an immune response. As mentioned previously,the apoEp1.B peptide comprises amino acids 239-252 of full lengthapolipoprotein E (apoE) including fragments, elongations, analogs andderivatives of the peptide. The inventors have shown that apoEp1.B butnot apoE or a peptide comprising amino acids 237-250 of apoE can inducethe activation and differentiation of immune cells.

Accordingly, the present invention provides a method of immunemodulation comprising administering an effective amount of an apoEp1.Bpeptide or a nucleic acid encoding an apoEp1.B peptide to a cell oranimal in need thereof.

The apoEp1.B peptide can be used to induce immune tolerance. Inparticular, the present inventors have demonstrated that the apoEp1.Bpeptide can activate immature cells to differentiate into tolerogenicdendritic cells. Accordingly, the present invention provides a method ofinducing immune tolerance comprising administering an effective amountof an apoEp1.B peptide or a nucleic acid encoding an apoEp1.B peptide toa cell or animal in need thereof. The induction of tolerogenic dendriticcells can have a wide variety of therapeutic applications includinginflammation, autoimmune disease and transplantation. The tolerogenicdendritic cells can be induced in vitro and then transferred to arecipient requiring the cells. Alternatively, the tolerogenic cells maybe directly induced in vivo.

The inventors have shown that the apoEp1.B peptide is useful ininhibiting inflammation. Accordingly, the present invention provides amethod for inhibiting inflammation comprising administering an effectiveamount of an apoEp1.B peptide or a nucleic acid encoding an apoEp1.Bpeptide to a cell or an animal in need thereof. In a preferredembodiment, the peptide can inhibit athersclerotic plaque formation invivo. Other inflammatory diseases that may be treated using the apoEp1.Bpeptide or nucleic acid encoding the apoEp1.B peptide include,arthritis, inflammatory bowel disease (IBD), Sjogren's syndrome,atherosclerosis, restenosis, transplant rejection, transplantvasculopathy, asthma, acute respiratory distress syndrome, allergy,psoriasis, multiple sclerosis, systemic lupus, acute glomerulonephrihs,spinal cord trauma, and others.

In a further embodiment, the apoEp1.B peptide can be used to prevent anautoimmune reaction or disease. The present inventors have demonstratedthat the apoEp1.B peptide can protect NOD mice from developing diabetes.Accordingly, the present invention provides a method for treating orpreventing an autoimmune disease comprising administering an effectiveamount of an apoEp1.B peptide or a nucleic acid encoding an apoEp1.Bpeptide to an animal in need thereof. In a preferred embodiment, theautoimmune disease is diabetes. Other autoimmune diseases which may betreated using the apoEp1.B peptide or nucleic acid encoding the apoEp1.Bpeptide include multiple sclerosis, EAE which is the mouse model ofmultiple sclerosis, rheumatoid arthritis, Sjogren's syndrome, lupus(SLE), autoimmune thyroid disease and others.

In a further aspect, the apoEp1.B peptide can be used to induce animmune response by activating immune cells. Accordingly, the presentinvention provides a method of inducing an immune response comprisingadministering an effective amount of an apoEp1.B peptide or a nucleicacid encoding an apoEp1.B peptide to an animal in need thereof. In oneembodiment, the peptide, in combination with other cytokines such asIL-4, GM-CSF, TNFα and Flt3 ligand induce immature dendritic cells todifferentiate into mature immunogenic dendritic cells. Mature dendriticcells can be used in a wide variety of applications including tumorimmunotherapy.

In another aspect, the apoEp1.B peptide can be used to treat tumors ofimmune origin by inducing their differentiation. In particular, thepresent inventors have demonstrated that the apoEp1.B peptide can inducethe differentiation and activation of monocytic, monoblastic leukemiaand lymphoma tumor cells. Accordingly, the present invention provides amethod of treating a tumor comprising administering an effective amountof an apoEp1.B peptide or a nucleic acid encoding an apoEp1.B peptide toan animal in need thereof.

The apoEp1.B peptide or nucleic acid encoding the apoEp1.B peptide mayalso be used to treat or prevent other diseases or conditions requiringimmune activation (including infectious diseases such as viralinfections) and immune tolerance (including tissue or organtransplantation, allergies and the above noted inflammatory andautoimmune diseases).

Administration of an “effective amount” of the apoEp1.B peptide ornucleic acid of the present invention is defined as an amount effective,at dosages and for periods of time necessary to achieve the desiredresult. The effective amount of an compound of the invention may varyaccording to factors such as the disease state, age, sex, and weight ofthe animal. Dosage regima may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. The term“animal” as used herein includes all members of the animal kingdomincluding humans.

In all of the above described therapeutic methods, the apoEp1.B peptidemay be administered in vivo or ex vivo. In ex vivo applications, theapoEp1.B peptide may be administered to cells that have been removedfrom the patient in an in vitro culture. After incubating the cells andpeptide for a period of time sufficient for the desired effect, thecells may be re-introduced into the patient's body. In one example,monocytes may be removed from a patient and cultured with apoEp1.B toallow them to mature. In addition, tumor antigens or autoantigens may beadded when treating cancer or autoimmune diseases, respectively. Themature monocytes expressing antigen can be re-introduced into thepatient and will induce an immune response to the tumor or autoantigen.

B. Pharmaceutical Compositions

The present invention includes pharmaceutical compositions containingthe apoEp1.B peptide or nucleic acid or substances which modulate theeffects of apoEp1.B for use in the described methods for modulating theimmune response.

Such pharmaceutical compositions can be for intralesional, intravenous,topical, rectal, parenteral, local, inhalant or subcutaneous,intradermal, intramuscular, intrathecal, transperitoneal, oral, andintracerebral use. The composition can be in liquid, solid or semisolidform, for example pills, tablets, creams, gelatin capsules, capsules,suppositories, soft gelatin capsules, gels, membranes, tubelets,solutions or suspensions. The apoEp1.B peptide is preferably injected ina saline solution either intravenously, intraperitoneally orsubcutaneously.

Several modes of administration are available when using a compositioncontaining a nucleic acid molecule encoding an apoEp1.B protein.Recombinant molecules comprising an nucleic acid sequence encoding anapoEp1.B protein, or fragment thereof, may be directly introduced intocells or tissues in vivo using delivery vehicles such as retroviralvectors, adenoviral vectors and DNA virus vectors. They may also beintroduced into cells in vivo or in vitro using physical techniques suchas microinjection and electroporation or chemical methods such ascoprecipitation and incorporation of DNA into liposomes. Recombinantmolecules may also be delivered in the form of an aerosol or by lavage.The nucleic acid molecules of the invention may also be appliedextracellularly such as by direct injection into cells.

The pharmaceutical compositions of the invention can be intended foradministration to humans or animals. Dosages to be administered dependon individual needs, on the desired effect and on the chosen route ofadministration.

The pharmaceutical compositions can be prepared by per se known methodsfor the preparation of pharmaceutically acceptable compositions whichcan be administered to patients, and such that an effective quantity ofthe active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985).

On this basis, the pharmaceutical compositions include, albeit notexclusively, the active compound or substance in association with one ormore pharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and iso-osmotic with thephysiological fluids. The pharmaceutical compositions may additionallycontain other agents to enhance the efficacy of the apoEp1.B peptide ornucleic acid.

C. Peptide Mimetics

The present invention also include peptide mimetics of the apoEp1.Bpeptides of the invention. For example, a peptide derived from a bindingdomain of apoEp1.B will interact directly or indirectly with anassociated molecule in such a way as to mimic the native binding domain.Such peptides may include competitive inhibitors, enhancers, peptidemimetics, and the like. All of these peptides as well as moleculessubstantially homologous, complementary or otherwise functionally orstructurally equivalent to these peptides may be used for purposes ofthe present invention.

“Peptide mimetics” are structures which serve as substitutes forpeptides in interactions between molecules (See Morgan et al (1989),Ann. Reports Med. Chem. 24:243-252 for a review). Peptide mimeticsinclude synthetic structures which may or may not contain amino acidsand/or peptide bonds but retain the structural and functional featuresof a peptide, or enhancer or inhibitor of the invention. Peptidemimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc.Natl. Acad, Sci USA 89:9367); and peptide libraries containing peptidesof a designed length representing all possible sequences of amino acidscorresponding to a peptide of the invention. Peptide mimetics may bedesigned based on information obtained by systematic replacement ofL-amino acids by D-amino acids, replacement of side chains with groupshaving different electronic properties, and by systematic replacement ofpeptide bonds with amide bond replacements. Local conformationalconstraints can also be introduced to determine conformationalrequirements for activity of a candidate peptide mimetic. The mimeticsmay include isosteric amide bonds, or D-amino acids to stabilize orpromote reverse turn conformations and to help stabilize the molecule.Cyclic amino acid analogues may be used to constrain amino acid residuesto particular conformational states. The mimetics can also includemimics of inhibitor peptide secondary structures. These structures canmodel the 3-dimensional orientation of amino acid residues into theknown secondary conformations of proteins. Peptoids may also be usedwhich are oligomers of N-substituted amino acids and can be used asmotifs for the generation of chemically diverse libraries of novelmolecules.

Peptides of the invention may also be used to identify lead compoundsfor drug development. The structure of the peptides described herein canbe readily determined by a number of methods such as NMR and X-raycrystallography. A comparison of the structures of peptides similar insequence, but differing in the biological activities they elicit intarget molecules can provide information about the structure-activityrelationship of the target. Information obtained from the examination ofstructure-activity relationships can be used to design either modifiedpeptides, or other small molecules or lead compounds which can be testedfor predicted properties as related to the target molecule. The activityof the lead compounds can be evaluated using assays similar to thosedescribed herein.

Information about structure-activity relationships may also be obtainedfrom co-crystallization studies. In these studies, a peptide with adesired activity is crystallized in association with a target molecule,and the X-ray structure of the complex is determined. The structure canthen be compared to the structure of the target molecule in its nativestate, and information from such a comparison may be used to designcompounds expected to possess desired activities.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1 ApoEp1.B Induces Dendritic Cells and Differentiationof Tumor Cells

Materials and Methods

Reagents

Phorbol, 12-myristate, 13-acetate (PMA) (Calbiochem, La Jolla, Calif.)was dissolved in ethanol and stored at −80° C.

Mice

C57BL/6J(B6) (H2b), apoE knockout (H2b), and BALB/c (H2d) mice between 8to 18 weeks used in this study were purchased from Jackson Laboratories,Bar Harbor, Me. Mice were fed a regular mouse chow (#5012), low in fat(4.5% wt/wt) and cholesterol (0.022% wt/wt) (Ralston, Purina, St. Louis,Mo.).

Cells and Culture Medium

Murine monocytic PU5-1.8 and J77A1.4 transformed cell lines, humanmonoblastic leukemia THP-1 and U-937, and murine B cell lymphoma A20cells were cultured in RPMI 1640 medium (Gibco Laboratories, GrandIsland, N.Y.) containing 5×105 M 2ME, 10 mM HEPES, 2 mM glutamine, 5.0IU/ml penicillin streptomycin (Gibco) and 10%. heat inactivated FetalBovine Serum (FBS) (Hyclone Laboratories Inc., Logan, Utah)(cRPMI).

Peptide Synthesis and Purification and Proteins

Peptides were synthesized on a Beckman 990C peptide synthesizer aspreviously described (MacNeil et al., 1993). Peptides were then purifiedby HPLC on a reverse phase C18 column with wateracetonitrile gradient.Peptides were dissolved at 2 mg/ml in distilled H₂O and filtersterilized through a 0.22 μm filter and further diluted in either cRPMI1640 before use in proliferation assays, or emulsified in CFA or IFA forimmunization of mice. Human plasma VLDL purified apoE (Calbiochem) wasused as native apoE.

Single Cell Suspension Preparation

Mice were euthanized in a CO₂ chamber and either spleens or lymph nodeswere removed aseptically and immersed in ice-cold PBS. Cells wereseparated by mincing tissues through a fine-mesh sieve. The cells werethen pelleted by centrifugation at 1500 rpm for 5 min and supernatantdiscarded. Erythrocytes in spleen preparations were lysed using ACKlysis buffer (0.15M NH₄Cl, 1.0 mM KHCO₃, 0.1 mM Na₂EDTA, pH 7.3) for 2minutes at room temperature and then resuspended in PBS and pelletedagain twice to wash cells.

T Cell Proliferation Assay

BALB/c mice were injected under light anesthesia with 50 μl volumescontaining 0, 1, 10 or 100 μg/ml of apoE peptide emulsified in IFA orCFA in the hind footpad. After 10 days draining (popliteal) lymph nodeswere removed and T cells were purified by passage through nylon wool.5×10⁵ purified T cells and 2×10⁵ gamma-irradiated (⁶⁰Co, 2500rads)(Atomic Energy, Canada) autologous spleen APC's were incubated with0, 1, 10, or 100 μg/ml apoE peptide in 96 well flat bottom microtitreplates at 5% CO₂, 37° C. for 3 days. PPD (20 μg/ml) served as a positivecontrol where CFA was used. Either APC's or T cells alone incubated witheither stimulating peptide served as negative controls. 50 μl [³H]-TdR(0.5 μCi/well) was added for an additional 18 hours and then cells werethen harvested (Tomtec, Orange, Conn.). [³H]-TdR incorporation wasmeasured on a Microbeta Liquid Scintillation Counter (Wallac, Turku,Finland).

Proliferation Assays

Unprimed spleen or peritoneal exudate cells (PEC's) were incubated with0, 1, 10, or 100 μg/ml apoEp1.B or negative control in 96 well flatbottom microtitre plates at 5% CO₂, 37° C. for 2 days. 50 μl [³H]-TdR(0.5 μCi/well) was added for an additional 18 hours and then cells werethen harvested (Tomtec, Orange, Conn.). [³H]-TdR incorporation wasmeasured on a Microbeta Liquid Scintillation Counter (Wallac, Turku,Finland). PU5-1.8, J77A1.4, A20, and U-937 cell lines were treatedsimilarly, but incubated with peptide for 12 hours prior to addition of[³H]-TdR for an additional 18 hours. Results from U-937 cell lines arepresented in FIG. 15. Cell supernatants were harvested from most cellcultures before radioisotype addition and assayed for cytokine content.

Mixed Leukocyte Reaction Assays

Primary allogeneic MLR was set up with NOD splenocytes as stimulatorsand nylon wool enriched BALB/c naive T cells as responders. Stimulatorcells were pre-incubated for 3 days with different combinations ofapoEp1.B, GM-CSF, and PU5-1.8 supernatant containing TNF. Stimulatorcells were then treated with mitomycin C (50 μg/ml, 20 min at 37° C.;Sigma) and co-cultured with 4×10⁴ responder for 3 days at which timecells were pulsed with 50 μl [³H]-TdR (0.5 μCi/well) for 24 hr. Cellswere harvested (Tomtec) and [³H]-TdR incorporation was measured on aMicrobeta Liquid Scintillation Counter (Wallac). Each bar represents themean cpm from triplicate cultures.

Cytokine Analysis

Culture supernatants were tested for IL-2, IL-4 and IFN_(γ)concentration using sandwick ELISA assays. Manufacturer's (Pharmingen)protocols were followed. 96-well microtitre ELISA plates were coatedwith 50 μl, 1 μg/ml α-IL-2, α-IL-4 or α-IFN_(γ) mAB in coating buffer(0.1 M NaHCO₃, pH 8.2) overnight at 4° C. Plates were washed twice withPBS-T (PBS, Tween-20 (Sigma, St. Louis, Mo.) and plates were blocked for2 hr at room temperature with 100 μl blocking buffer (PBS, 5% BSA).Plates were washed twice and 50 μl samples were added in triplicate foran overnight, 4° C. incubation. Plates were then washed 5 times andfollowing a 2 hr room temperature incubation period with 50 μl, 1 μg/mlbiotinylated α-IL-2, α-IL-4 or α-IFNγ mAB (Pharmingen), plates werewashed 8 times. 50 μl, 1 μg/ml streptavidin-alkaline phosphatase(Pharmingen) was then added for 1 hr at room temperature. Following 8washes, 50 μl p-nitrophenyl phosphate substrate (pNPP (Sigma)) was addedand absorbance determined at O.D. 405 nm using a microplate reader(Biorad, Hercules, Calif.) after colour had developed.

Immunofluorescence and Flow Cytometry

1×10⁶ cells were washed with ice cold PBS and resuspended in 25 μl PBSand incubated with 10 μl normal mouse serum and fluorochrome conjugatedanti-mouse D11a, CD11b, CD14, MHC class II, CD95, CD62L, CD62E, B7-1,B7-2 and ICAM-1 Pharmingen, Mississauga, Canada) monoclonal antibodies(mAb's) for 45 minutes on ice. endritic cell markers DEC-205, 33D1,CD11c (N418) unconjugated antibodies were imilarly treated, however,following primary antibody incubation,cells were washed 3 times and asecondary goat anti-rat (for 33D1 and DEC-205 ab's) and goatanti-hamster (for CD11c ab) fluorochrome-conjugated antibody was thenadded for 45 min on ice. Cells were then washed three times. All sampleswere resuspended in 300 μl PBS and the fluorescence of stained cells wasanalyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View,Calif.) and data collected was analyzed using Lysis II(Becton-Dickinson) software.

Cell Cycle Analysis

1×10⁶ cells were washed and resuspended in hypotonic propidium iodide(PI) staining solution (0.1% [w/v] sodium citrate, 0.1[v/v] TritonX-100, 0.05 μg/ml propidium iodide) and left to stain in the darkovernight at 4° C. Cells were analyzed on a FACScan flow cytometer andthe percentage of cells in each phase of the cell cycle were quantitatedusing ModFit software (Becton Dickinson). Aggregates were excluded fromthe analysis with the use of the doublet discrimination module andsubsequent gating on the linear red (FL2) fluorescence area and widthparameters.

Intraperitoneal Injection

BALB/c mice were injected i.p. with 300 μg apoE peptide and 48 hourslater PECs were collected post-mortem by peritoneal lavage using icecold saline.

Adoptive Transfer Studies

RBC-depleted diabetogenic splenocytes from 17-20 wk-old female NOD micewere resuspended in saline. 8 to 10 wk old NOD.SCID mice were injectedi.p. with 0.2 μl saline containing 10⁷ of the diabetogenic splenocytes.These same mice were then immunized with either 200 μg/ml apoEp1.B or200 μg/ml apoEp2 via the footpad. Urine glucose levels were monitoredbiweekly using glucose enzymatic test strips (Eli Lilly, Toronto, Ont.).Once urine tested positive, then blood glucose levels (BGL) were testedusing Glucometer Encore (Miles/Bayer). Mice exhibiting BGL >11.1 mmol/L(200 mg/dl) for 2 consecutive weeks were considered diabetic.

RESULTS ApoEp1.B Peptide Induces Dendritic-like Cell Morphology

Unprimed splenocytes, enriched spleen monocytes and monocytic PU5-1.8and J77A4.1 cell lines from BALB/c mice were incubated with 0, 1, 10, or100 μg/ml apoEp1.B (239-252) or negative control apoEp1.D (236-249)peptide to assess activity. After only 2 hr, apoEp1.B, but notapEp1.D-incubated cells started to detach from plastic plates andaggregate in suspension optimally at 100 μg/ml. After 24 hr,morphologically these cells appeared less rounded, more granular.Photographs were taken after 24 hours, the results are shown in FIG. 1(magnification 400×). At 48 hours they displayed dendritic-likeprocesses. FACS analysis confirmed an increase in cell size andgranularity (FIG. 2).

ApoEp1.B Peptide Induces DC-like Marker Expression

Morphologically apoEp1.B-treated cells appeared DC-like. This wasconfirmed this by FACS analysis. Unprimed BALB/c splenocytes and PU5-1.8cells were incubated for 24 hr with 100 μg/ml apoEp1.B or controlpeptide apoEp1.D and then stained for FACS analysis. Surface expressionof CD14, CD11a, CD11b, CD11c, B7-1, B7-2, DEC-205, 33D1, MHC class 11,fas, CD40, CD62L (L-selectin) and CD43 (leukosialin) were increasedabove background on apoEp1.B-incubated PU5-1.8 cells after 24 hrincubation (FIGS. 3 and 4). With repeated experimentation, expression ofMac-3 and CD54 (ICAM-1) were found to be relatively variable.

To assess whether apoEp1.B-induced phenotypic changes aredose-dependent, 10⁵ PU5-1.8 cells were incubated for 20 hr with 0(medium), 1, 10 or 100 μg/ml apoEp1.B in 24-well plates and were thenstained for FACS analysis. 10,000 events were collected. B7-1 marker isshown. The results, shown in FIG. 5 illustrate that 100 μg/ml apoEp1.Bwas found to be optimal, however, 1 μg/ml resulted in slight phenotypicchanges.

Surface expression of B7-1, B7-2, MHC class 11, CD28, CD11a, CD11c and33D1 were increased above background on apoEp1.B-incubated splenocytesafter 24 hr incubation (see FIG. 4). There was also an increase in CD40and a decrease in L-selectin (data not shown) on apoEp1-treatedsplenocytes. There were no changes in the expression of CD16/32 at anytime.

ApoEp1.B Peptide-induced Differentiation and Activation is not Strain orSpecies-specific

Unprimed B6 and NOD splenocytes were incubated with 100 μg/ml apoEp1.Bor apoEp1.D to determine whether apoEp1.B was strain-specific. After 48hr in culture, cells were stained for FACS analysis. B6 and NODsplenocytes responded to apoEp1.B with similar marker expressionincrease to that of BALB/c splenocytes (data not shown). Naive spleencells from apoE deficient (apoE K.O.) mice were also tested to determinewhether in vivo production of apoE had any influence on this effect.ApoE K.O. splenocytes also responded with a similar marker expressionincrease to that of BALB/c (data not shown). Since human apoEp1.B(HapoEp1.B) (239-252) peptide has a high degree of homology to murineapoEp1.B (²³⁹T to ²³⁹A and 248I to ²⁴⁸A), species cross-reactivity wastested. Human monocytic cell line U-937 responded within 24 hr to 100μg/ml apoEp1.B with similar, but less profound morphological andphenotypic changes to that of PU5-1.8 cells (FIG. 15). To determinewhether the HapoEp1.B had a similar effect on murine cells, 10⁵ PU5-1.8cells were incubated with 100 μg/ml HapoEp1.B or murine apoEp1.B for 24hr. The cells were then stained for FACS analysis. HapoEp1.B inducedcell clustering and an increase in similar markers to that of murineapoEp1.B, but to a lesser degree (B7-1 marker expression shown in FIG.6).

ApoEp1.B Induces DC-like Cells When Injected Intraperitoneally

To assess whether apoEp1.B could induce DC-like cells in vivo, BALB/cmice were injected i.p. with 300 μg/ml apoEp1.B or apoEp1.D. After 48 hrPEC's were stained for FACS analysis. ApoEp1.B induced an increase incell size and granularity (FIG. 8A) and an increase in surfaceexpression of DEC-205, 33D1, CD11c, B7-1 and B7-2 (FIG. 8B). Unlabelledpeaks correspond to apoEp1.D. Mean fluorescent intensity shown inbrackets. The results demonstrate that apoEp1.B induces differentiationand activation of PECs in vivo similar to the in vitro system.

To assess functional activation of apoEp1.B treated cells, BALB/cunprimed splenocytes were incubated with or without apoEp1.B (100μg/ml)in vitro for 24 hours. Cells (washed and unwashed) were stimulated withConA for 12 hours and 3H was then added for an additional 12 hours.Cells were then harvested and readioactivity uptake counted. Results arepresented as the mean of triplicate wells (cpm+/−SEM). As shown in FIG.9, apoEp1.B treated cells respond to mitogen 3-fold higher thanuntreated cells.

Cytokines Produced After ApoEp1.B Immunization

ApoEp1.B or apoEp1.D-imrnmunized NOD lymph node cells were cultured for48 hr in the absence of challenge antigen or exogenous cytokine. Cellsupernatants were harvested and tested for cytokine content to assesswhether apoEp1.B induced a Th1 or Th2 response. ApoEp1.B-primed cellssecreted higher levels of IL-4 and lower levels of IL-2 than apoEp1.Dprimed cells (FIG. 12). There was no change in IFNγ.

T Cell Allostimulatory Activity of ApoEp1.B-induced DC

To measure the T cell allostimulatory or inhibitory ability of theseDC's, 4×10⁴ enriched BALB/c naive T cells were co-cultured with 40, 400,4000 and 40,000 mitomycin C-treated NOD splenocytes which had beenincubated with various stimualants for 3 d. T cell proliferation wasmeasured following 3 days of co-culture by [³H]-TdR incorporation. Theresults are shown in FIG. 12. Bars represent the mean of triplicatewells (cpm+/−S.D.). * and ** represent p<0.05 and p<0.001, respectivelyin 2-way ANOVA's compared to medium alone (nil) controls.ApoEp1.B-treated APC allostimulation was similar to that of untreatedAPC's at 4×10⁴ APC's per well concentration, however, there was aslightly higher level of apoEp1.B-treated APC-T cell stimulation overuntreated APC's at 4×10³ and 4×10² APC's. There was a dramatic increasein allostimulation when APC's were pre-incubated with GM-CSF,particularly at 4×10³ APC's per well. This increase was slightly, butsignificantly reduced when APC's were pre-incubated with GM-CSF plusapoEp1.B. The more striking result is depicted in FIG. 5B. APC's (4×10³)in all bars (except “T alone”) have been untreated (nil) orpre-incubated with 100 μg/ml apoEp1.B, apoEp1.B+GM-CSF, or GM-CSF alonefor 3 days prior to co-culture. Upon combining responder T cells withAPC, various exogenous factors were added, including 20 μg/ml Con A and5 μg/ml apoEp1.B. Very little proliferation was detected in wellscontaining T cells alone or APC alone. T cells proliferated well inresponse to Con A, however, when apoEp1.B was added, T cellallostimulation was completely abrogated.

DISCUSSION

It is known that monocytic cells can differentiate into DC's, however,GM-CSF has been long been thought to be essential for this effect(Scheicher et al., 1992). It has been shown here that a novel selfpeptide, apoEp1.B, alone induces DC's in vitro from both naive monocyticcells, and a murine cell line. These DC's possess the morphology andcell surface markers of classical DC's. ApoEp1.B also induces DC's invivo upon apoEp1.B immunization. These DC's may favour a Th2 response asshown by an increase in IL-4 and a decrease in IL-2 production, and maytherefore, be involved in split tolerance.

ApoEp1.B induced DC morphology of naive BALB/c splenocytes, enrichedspleen monocytes (data not shown), monocytic cell lines PU5-1.8 (FIG. 1)and J77A4.1 in vitro (data not shown). Following only 2 hr incubation,apoEp1.B-treated cells started to detach from plastic plates andaggregated in suspension, a process characteristic of DC's. Since therewas increased cell death, proliferation assays did not show an overallincrease in cell number (data not shown). This is consistent with theloss of proliferation characteristic of cell differentiation.

ApoEp1.B-treatment of PU5-1.8 cells induced an increase in DC-specificmarkers, DEC-205, 33D1 and CD11c. Other markers that consistentlyincreased were CD11a, B7-1, B7-2, MHC class II, fas, CD40 andL-selectin.

ApoEp1.B stimulation of splenocytes induced an increase in CD40, CD11a,CD11c, 33D1, B7-1, B7-2, MHC class II, fas and fasL expression at 24 hr.A similar profile was recorded following 72 hr post-apoEp1.B treatment(data not shown). These changes were not as dramatic as apoEp1.B-inducedPU5-1.8 surface marker changes. This does not detract from the resultsobtained with PU5-1.8 cells as these cells are transformed and capableof rapid growth, cell division and protein synthesis.

Changes in B7 costimulatory molecules would inevitably alter T cellresponses. Furthermore, an increase in MHC class II enables DC's topresent peptides at a higher density which renders them more efficientat either T cell activation or tolerance. An increase in expression ofadhesion molecules such as LFA-1 may enable DC's migration from varioustissues to lymphoid organs where they present captured Ag.

In contrast to splenocytes, L-selectin expression on PU5-1.8 cellsincreases with apoEp1.B incubation (data not shown). L-selectin israpidly downregulated on activated cells and thus the spleen cell datasuggests that apoEp1.B activates these cells. These conflicting resultsare not extraordinary when considering that PU5-1.8 cells aretransformed as well as a homogenous population. Heterogeneous cell-cellinteractions as well as paracrine stimulatory and inhibitory factorsthat affect immune responses may explain some of the differences betweenspleen and PU5-1.8 cells. Furthermore, transformed PU5-1.8 cells maylack mechanisms necessary for L-selectin downregulation, such asspecific proteases for L-selectin cleavage. CD28 expression onsplenocytes is also increased slightly with apoEp1.B treatment.

A slight increase in macrophage differentiation markers CD14 and Mac-3,indicates that maturation of macrophages may be also occurring inapoEp1.B-treated cultures. Whether these cells then develop into DC'S,apoptose or remain as macrophages is unknown.

Following i.p. injection, apoEp1.B induced an increase in PEC size andgranularity and an increase in DC-specific markers, DEC-205, 33D1 andCD11c as well as B7-1 and B7-2. Therefore, injection of apoEp1.B i.p.induces PEC's to express a similar DC-like phenotype compared to invitro experiments. The increase in DC marker expression is not aspronounced as it was in vitro, probably due to the dilution and/orclearance of apoEp1.B peptide in vivo. However, since DC's are efficientat T cell stimulation or inhibition, even a modest increase in numbermay have a profound effect on an immune response.

The treatment of NOD mice with apoEp1.B stimulated an increase in IL-4secretion, presumably by Th2-like cells and a decrease in IL-2. Theimmunization of mice with apoEp1.B also induced an increase in DC's.

Whether apoEp1.B is a naturally cleaved product of apoE is unclear butdoubtful. ApoEp1.B binds I-A^(d) with a similar affinity to that of. theoriginal apoEp1 peptide (which does not induce DC's), yet was not elutedin elution experiments, it is therefore, probably not naturally cleaveda high levels.

Here we show that apoEp1.B, stimulates mo/ma activation. In addition,apoEp1.B induces DC's that may favour a Th2-like response. These DC'smay be partially activated or not fully mature, fully activated orinduced to full maturity by inflammatory signals.

Example 2 ApoEp1.B Inhibits Atherosclerotic Plaque Development

To investigate the role of apoEp1.B peptide in inflammation,particularly in response to arterial injury, a balloon angioplastyinjury model which has been well characterized by Dr. Alex Lucas (Lucaset al. 1996 Circ. 94:2898-900) was used. 300 μg/ml of apoEp1 or apoEp1.Bpeptide were infused intra-arterially immediately prior to angioplastyat the site of injury in the iliofemoral artery of the rat. Positivecontrol SERP-1 and negative control saline alone were also used. Therats were monitored carefully and euthanized 6 weeks after treatment andiliofemoral arteries were assessed for plaque development byhistochemistry. Careful measurement of arterial thickness and lumen sizeshowed apoEp1.B (500 μg) peptide reduced lesion size in this model. Infact, in many of the apoEp1.B treated rats, no plaque was detected. Noadverse effects were observed in apoEp1.B treated rats. The results arepresented in Table 3 and FIG. 10.

Example 3 ApoEp1.B Immunization Protects Mice From Diabetes

8 female 8 week old NOD mice were immunized with 250 μg apoEp1.B peptideemulsed in IFA in one footpad. 6 negative control mice were immunizedwith saline in IFA. Mice were monitored for a following 4 months andtheir urine glucose tested to assess diabetes development. At the 8month end date, all 8 apoEp1.B immunized mice still remain diabetesfree. 4 of the 6 mice treated with saline in IFA are deceased due todiabetes. The results, shown in FIG. 14, demonstrate that apoEp1.Bimmunization protects diabetes prone mice.

Example 4 ApoEp1.B-immunization Induces Th2-like Cells

NOD mice were immunized via the footpad with 200 μg/ml apoEp1.B orapoEp1 D peptide in IFA. Draining lymph nodes were removed following 7days and cells cultured in 96-well microtiter plates in the absence offurther stimulation. Cell culture supernatants were harvested at 48 hrand tested for cytokine content. The results, shown in FIG. 11,demonstrate that apoEp1.B immunization induce Th2-like cells. (Resultsof IL-2, IL-4 and IFNγ are presented as the mean of triplicate wells(O.D.+/−SEM). * represents p<0.001 in 2-way ANOVA's compared toapoEp1.D-treated controls.)

To determine whether the Th2 response induced by apoEp1.B immunizationinfluenced IDDM incidence, 10 female 8 week old NOD mice were immunizedwith 200 μg/ml apoEp1.B and 10 mice with negative control apoEp2 emulsedin IFA in one footpad. Mice were monitored for the following 8 monthsand their urine and blood glucose tested for disease onset. At 10months, 7 of 10 apoEp1.B-immunized mice remain diabetes free, whereas, 1of 10 apoEp2-treated mice remaines disease-free. (FIG. 13A).

Example 5 ApoEp1.B Immunization Protects NOD.SCID Mice From AdoptivelyTransferred Diabetes

To test the protective effect of apoEp1.B in an alternative model ofIDDM, 10⁷ diabetogenic splenocytes were adoptively transferred to 20NOD.SCID mice. 10 of these mice were immunized with 200 μg/ml apoEp1.B,the other with 200 μg/ml apoEp2, the other with 200 μg/ml apoEp2. All 10negative controls have succumbed to disease after 5 weeks. 5 of the 10apoEp1.B mice remain diabetes-free at 9 weeks (FIG. 13B).

Discussion of Results From Examples 3,4 and 5

Since a Th2 response can be protective in the NOD model of IDDM andsince apoEp1.B immunization of NOD mice favours a Th2 type response (seeFIG. 11), apoEp1.B was tested for disease protection in these mice.ApoEp1.B/IFA immunization significantly delayed disease onset in NODmice and in adoptively-transferred NOD.Scid mice. ApoEp1.B-protectedmice had slightly less islet infiltration as compared to unprotectedmice (data not shown), indicating that apoEp1.B protection may involvethe induction of regulatory cells rather than deletion of destructiveeffector cells.

APC's from NOD mice are thought to have functional and/ordifferentiation defects (Serreze et al., 1988, Serreze et al., 1993).Since NOD Th2 cells are more dependent on B7 costimulation than Th1cells (Rulifson et al., 1997), a proinflammatory Th1 response maypredominate in these mice by default. Consistent with this are studiesdemonstrating that Th2 cell hyporesponsiveness intrinsic to NOD mice(Zipris 1991a and 1991b) is reversible either by IL-4 (Rapoport et al.,1993, Mueller et al., 1996, Cameron et al., 1997) or CD28 (Arreaza etal., 1997) administration in vivo. Anti-CD28 treatment is thought topromote Th2 survival and expansion. Furthermore, costimulation deficientmice (CD28−/−) display more severe IDDM (Rulifson et al., 1997).

It is unclear what role DC's play in IDDM, however, the importance ofDC's in disease is emphasized by the DC K.O. mouse (RelB−/−). These micedisplay aggressive multi-organ infiltration and inflammation. Anothergroup reported that pancreatic DC's can induce disease protection upontransfer (Clare-Salzler et al., 1992). These findings suggest that DC'smay play a protective role in IDDM. Furthermore, Voorbij et al. (1989)showed that DC's were the first cells to accumulate around pancreaticislets in the spontaneously diabetic BB rat model, followed bylymphocytes.

Whether the induction of DC's upon apoEp1.B injection has a directeffect on disease protection is unknown, however, the resulting Th2response may be mediating protection. It is proposed that apoEp1.Bimmunization stimulates DC differentiation with an increase in B7costimulatory molecules. These more efficient APC's, capable of strongercostimulation, rescue IL-4 producing Th2 cell responsiveness indiabetes-prone mice. This in turn reduces disease incidence.Alternatively or additionally, apoEp1.B may act directly on T cells,increasing CD28 expression and/or lowering the threshold forstimulation. Since Th1 cells are already stimulated in NOD mice,protective Th2 or Th3 cell stimulation may be preferentially restored.Consistent with this statement is the data showing no change in IFNproduction, yet an increase in IL-4.

Since the lack of Th2 cell responsiveness intrinsic to NOD mice (Zipris1991a and 1991b) is reversible, it is proposed the apoEp1.B immunizationrescues Th2 responsiveness in diabetes-prone mice, which in turn reducesdisease incidence. Since T cells require APC's for stimulation, wespeculate that apoEp1.B stimulates a Th2-inducing APC, possible of theDC phenotype. Irrespective of mechanism, apoEp1.B may offer a potentialself peptide therapy for diabetes as well as other Th1-mediatedautoimmune diseases.

Example 6 Modifications to ApoEp1.B Sequence

Various amino acid substitutions and elongations were made to theapoEp1.B peptide as illustrated in Tables 1 and 2. The modified peptideswere incubated at 100 μg/ml with 10⁵ PU5-1.8 cells for 20 hours. Thecells were stained with B7-1 for FACS analysis. The results demonstratethat some, but not all, single a.a. substitutions, elongations ordeletions can decrease or abrogate apoEp1.B effects (see Table 1 and 2and FIGS. 16 and 18). This supports the notion that there is a receptorto which apoEp1.B binds and certain a.a. changes decreasepeptide-receptor affinity. The deletion of 239^(T) results in a greatlyreduced effect on surface marker changes. However, the substitution ofthis same a.a. with an alanine (as in the sequence of HapoEp1.B) hasonly a slightly reduced effect compared to apoEp1.B. Therefore, thelength of apoEp1.B is likely important in receptor binding. It alsoappears that the structural integrity resides in the carboxy terminalregion of the apoEp1.B peptide as even slight changes in that regiondecreases or abolishes the activity of the peptide.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1 ApoEp1.B Elongations and Truncations DC Marker Name SequenceResidues Changes apoEp1 EEQ TQQ IRL QAE IFQ AR (SEQ.ID.NO.:9) 236-252slight apoEp1.Ba  EQ TQQ IRL QAE IFQ AR (SEQ.ID.NO.:1O) 237-252 −apoEp1.Bb   Q TQQ IRL QAE IFQ AR (SEQ.ID.NO.:7) 238-252 ++ apoEp1.B    TQQ IRL QAE IFQ AR (SEQ.ID.NO.:1) 239-252 +++++ apoEp1.Bc      QQIRL QAE IFQ AR (SEQ.ID.NO.:8) 240-252 ++ apoEp1.Bd       Q IRL QAE LFQAR (SEQ.ID.NO.:11) 241-252 −

TABLE 2 ApoEpl.B Amino Acid Substitutions DC Marker Name SequenceResidues Changes apoEp1.B A²⁴⁰ TAQ IRL QAE IFQ AR (SEQ.ID.NO.:3) 239-252+++ apoEp1.B A²⁴¹ TQA IRL QAE IFQ AR (SEQ.ID.NO.:4) 239-252 +++++apoEp1.B A²⁴² TQQ ARL QAE IFQ AR (SEQ.ID.NO.:5) 239-252 ++ apoEp1.B A²⁴³TQQ IAL QAE IFQ AR (SEQ.ID.NO.:6) 239-252 +++ apoEp1.B A²⁴⁴ TQQ IRA QAEIFQ AR (SEQ.ID.NO.:12) 239-252 − apoEp1.B A²⁴⁵ TQQ IRL AAE IFQ AR(SEQ.ID.NO.:13) 239-252 − apoEp1.B A²⁴⁷ TQQ IRL QAA IFQ AR(SEQ.ID.NO.:14) 239-252 − apoEp1.B A²⁴⁸ TQQ IRL QAE AFQ AR(SEQ.ID.NO.:15) 239-252 − apoEp1.B A²⁴⁹ TQQ IRL QAE IAQ AR(SEQ.ID.NO.:16) 239-252 − apoEpl.B A²⁵⁰ TQQ IRL QAE IFA AR(SEQ.ID.NO.:17) 239-252 − apoEp1.B A^(239,240) AAQ IRL QAE IFQ AR(SEQ.ID.NO.:18) 239-252 − apoEp1.B A^(240,241) TAA IRL QAE IFQ AR(SEQ.ID.NO.:19) 239-252 − apoEp1.B A²³⁹⁻²⁴¹ AAA IRL QAE IFQ AR(SEQ.ID.NO.:20) 239-252 − human apoEp1.B AQQ IRL QAE AFQ AR(SEQ.ID.NO.:2) 239-252 ++++

TABLE 3 Plaque Lumen Area Thickness Plaque area (mean +/− S.E.) (mean+/− S.E.) (mean +/− S.E.) saline 0.027 +/− 0.012 0.139 +/− 0.028 0.059+/− 0.010 apoEp1.B 0.156 +/− 0.020 0.003 +/− 0.003 0.002 +/− 0.002P-value <.0001 <.0001 <.0001

FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

Arreaza, G. A., Cameron, J. J., Jaramillo, A., Gill, B. M., Hardy, D.,Laupland, K. B., Rapoport, M. J., Zucker, P., Chakrabarti, S., Chensue,S. W., Qin, H. Y., Singh, B., Delovitch, T. L. (1997) Neonatalactivation of CD28 signaling overcomes T cell energy and preventsautoimmune diabetes by an IL-4-depednent mechanism. J. Clin Invest. Nov1, 100(9):2243-53.

Cameron, M. J., Arreaza, G. A., Zucker, P., Chensue, S. W., Strieter, R.M., Chakrabarti, S., Delovitch, T. L. (1997) IL-4 prevents insulitis andinsulin-dependent diabetes mellitus in nonobese diabetic mice bypotentiation of regulatory T helper-2 cell function. J. Immunol, Nov.15, 159(10):4686-92.

Caux, C., Dezutter-Dambuyant, C., Schmitt, D., and Banchereau, J.(1992). GM-CSF and TNF-a cooperate in the generation of dendriticLangerhans cells. Nature 360, 258-261.

Caux, C., Massacrier, C., Vanbervliet, B., Dubois, B., Van Kooten, C.,Durand, I., and Banchereau, J. (1994). Activation of human dendriticcells through CD40 cross-linking. J. Exp. Med. 180,1263-1272.

Christianson, S. W., Shultz, L. D., and Leiter, E. H. (1993). Adoptivetransfer of diabetes into immunodeficient NOD-scid/scid mice—relativeconttributions of CD4+ and CD8+ T-cells from diabetic versus prediabeticNOD.NON-Thy-1a donors. Diabetes 42, 44-55.

Clare-Salzler, M. J., Brooks, J., Chai, A., Van Herle, K., and Anderson,C. (1992). Prevention of diabetes in nonobese diabetic mice by dendriticcell transfer. J. Clin. Invest. 90, 741-748.

Daniel, D., Gill, R. G., Schloot, N., and Wegrnann, D. (1995). Epitopespecificity, cytokine production profile and diabetogenic activity ofinsulin-specific T cell clones isolated from NOD mice. Eur J Immunol.25,1056-1062.

Delovitch, T. L., and Singh, B. (1997). The nonobese diabetic mouse as amodel of autoimmune diabetes: immune dysregulation get the NOD. Immunity7, 727-738.

Dyer, C. A., Smith, R. S., and Curtiss, L. K. (1991). Only multimers ofa synthetic peptide of apolipoprotein E are biologically active. J.Biol. Chem. 266, 15009-15015.

Fitch, F. W. McKisic, M. D., Lancki, D. W., and Gajewski, T. F. (1993).Differential regulation of murine T lymphocyte subsets. Annu RevInmmunol. 11, 29-48.

Finkelman, F. D., Lees, A., Bimbaum, R., Gause, W. C., and Morris, S. C.(1996). Dendritic cells can present antigen in vivo in a tolerogenic orimmunogenic fashion. J. Immunol. 157, 1406-1414.

Galy, A., Travis, M., Cen, D., and Chen, B. (1995). Human B, T, naturalkiller and dendritic cells arise from a common bone marrow progenitorcell subset. Immunity 3, 459-473.

Gao, J.-X., Madrenas, J., Zeng, W., Zhong, R., and Grant, D. (1997).Generation of dendritic cell-like antigen-presenting cells in long-termmixed leucocyte culture: phenotypic and functional studies. Immunology.91, 135-144.

Grosjean, I., Caux, C., Bella, C., Berger, I., Wild, F., Banchereau, J.,and Kaiserlian. (1997). Measles virus infects human dendritic cells andblocks their allostimulatory properties for CD4+ T cells. J. Exp. Med.186, 801-812.

Groux, H., Bigler, M., de Vries J. E., and Roncarolo,M. (1996).Interleukin-10 induces a long-term antigen-specific anergic state inhuman CD4+ T cells. J. Exp. Med 184: 19-29.

Haskins, K., and McDuffie, M. (1990). Acceleration of diabetes in youngNOD mice with a CD4+ islet-specific T cell clone. Science 249,1433-1436.

Hsieh, C.-S., Heimberger, A. B., Gold, J. S., O'Garra, A., and Murphy,K.M. (1992). Differential regulation of T helper phenotype developmentby interleukins 4 and 10 in alpha beta T cell-receptor transgenicsystem. Proc. Natl. Acad. Sci. USA 89, 6065-6069.

Hsu, F. J., Benike, C., Fagnoni, F., Liles, T. M., Czerwinski, D.,Taidi, B., Engleman, E. G., and R Levy. 1996. Vaccination of patientswith B-cell lymphoma using autologous antigen-pulsed dendritic cells.Nat. Med. 2:52-8.

Hunt, D., H. Michel, J. S. Dickinson, J. Shabanowitz, A. L. Cox, K.Sakaguchi, E. Appella, H. M. Grey, and A. Sette. 1992. Peptidespresented to the immune system by murine class II majorhistocompatibility complex IAd. Science 256:1817-1820.

Kalinski, P., Hilkens, C. M. U., Snijders, F., Snijdewint, F. G. M., andKapsenberg, M. L. (1997). IL-12-deficient dendritic cells, generated inthe presence of prostaglandin E2, promote type 2 cytokine production inmaturing human naive T helper cells. J. Immunol. 159, 28-35.

Katz, J. D., Benoist, C., and Mathis, D. (1995). T helper subsets ininsulin-dependent diabetes. Science 268, 1185-1188.

Kelly, M. E., M. A. Clay, M. J. Mistry, H. M. HsiehLi, and J. A. K.Harmony. 1994, Apolipoprotein E inhibition of proliferation ofmitogenactivated T lymphocytes: production of interleukin 2 with reducedbiological activity. Cell Immunol. 159:124-139.

Kuchroo, V. K., Das, M. P., Brown, J. A., Ranger, A. M., Zamvil, S. S.,Sobel, R. A., Weiner, H. L., Nabavi, N., and Glimcher, L. (1995). B7-1and B7-2 costimulatory molecules activate differentially the Th1/Th2developmental pathways: application to autoimmune disease therapy. Cell80, 707-718.

Liblau, R. S., Singer, S. M., and McDevitt, H. O. (1995). Th1 and Th2CD4+ T cells in the pathogenesis of organ-specific autoimmune diseases.Immunol Today 16, 34-38.

Lynch, D. H., Andreasen, A., Maraskovsky, E., Whitmore, J., Miller, R.,and Schuh, C. L. (1997). Flt3 ligand induces tumor regression andantitumor immune responses in vivo. Nat Med. 3, 625-631.

MacNeil, D., Fraga, E., and B. Singh. 1993. Characterization of murine Tcell responses to peptides of the variable region of self T cellreceptor chains. J. Immunol. 151:4045.

Mahley, R. W. 1988. Apolipoprotein E: cholesterol transport protein withexpanding role in cell biology. Science 240:622.

Maraskovsky, E., Brasel, K., Teepe, M., Roux, E. R., Lyman, S. D.,Shortman, K., and H. J. McKenna. 1996. Dramatic increase in the numbersof functionally mature dendritic cells in Flt3 ligand-treated mice:multiple dendritic dell subpopulations identified. J. Exp. Med.184:1953-1962.

Mayordama, J. I., Zorina, T., Storkus, W. J., Zitvogel, L., Celluzzi,C., Falo, L. D., Melief, C. J., Ildstad, S. T., Kast, W. M., Deleo, A.B., et. Al 1995. Bone marrow-derived dendritic cells pulsed withsynthetic tumour peptides elicit protective and therapeutic antitumourimmunity. Nat. Med. 12:1297-302.

Mosmann, T. R., and Coffman, R. L. (1989). TH1 and TH2 cells: differentpatterns of lymphokine secretion lead to different functionalproperties. Annu. Rev. Immunol. 7, 145-173.

Mueller, R., Krahl, T., and Sarvetnick, N. (1996). Pancreatic expressionof interleukin-4 abrogates insulitis and autoimmune diabetes in NODmice. J. Exp. Med. 184, 1093-1099.

Pennline, K. J., Roque-Gaffney, E., and Monahan, M. (1994). Recombinanthuman IL-10 prevents the onset of diabetes in the nonobese diabeticmouse. Clin. Immunol. Immunopathol. 71, 169-175.

Pepe, M., and L. Curtiss. 1986. Apolipoprotein E is a biologicallyactive constituent of the normal immunoregulatory lipoprotein, LDLIn. J.Immunol. 136:3716.

Peters, J. H., Gieseler, R., Thiele, B., and Steinbach, F. 1996.Dendritic cells: from ontogenetic orphans to myelomonocytic descendants.Immunol. Today. 6:273-278.

Powrie, F., and Coffman, R. L. (1993). Cytokine regulation of T-cellfunction: potential for therapeutic intervention. Imnmunol. Today 14,270-274.

Rapoport, M. J., Zipris, D., Lazarus, A. H., Jaramillo, A., Speck, E.,and Delovitch, T. L. (1993), IL-4 reverses thymic T cell proliferativeunresponsiveness and prevents diabetes in NOD mice. J. Exp. Med.178,87-99.

Rider B. J., Fraga, E., Yu Q. and Singh B. (1996). Immune responses toself-peptides naturally presented by murine class II majorhistocompatibility complex molecules. Mol. Immunol. 33, 625-633.

Romagnani, S. (1992). Human Th1 and Th2 subsets: regulation ofdifferentiation and the role in protection and immunopathology. Int.Arch. Allergy Immunol. 98, 279-285.

Romani, N., Gruner, S., Brang, D., Kampgen, E., Lenz, A., Trockenbacher,B., Konwalinka, G., Fritsch, P. O., Steinman, R. M., and Schuler, G.(1994). Proliferating dendritic cell progenitors in human blood. J. Exp.Med. 180, 83-93.

Rosenfield, M. E., Butler, S., Ord, V. A., Lipton, B. A., Dyer, C. A.,Curtiss, L. K., Palinski, W., and Witztum, J. L. (1993). Abundantexpression of apoprotein E by macrophages in human and rabbitatherosclerosis. Arterioscler. Thromb. 13, 1382-1389.

Rossini, A. A., Greiner, D. L., Friedman, H. P., and Mordes, J. P.(1993). Immunopathogenesis of diabetes mellitus. Diabetes Rev. 1, 43-75.

Rulifson, I. C., Sperling, A., Fields, P. E., Fitch, F. W., Bluestone,J. A. (1997) CD28 costimulation promotes the production of the Th2cytokines. J. Immunol., Jan. 15, 158(2):658-65.

Salomon, B., Cohen, J. L., Masurier, C., and Klatzmann, D. (1998). Threepopulations of mouse lymph node dendritic cells with different originsand dynamics. J. Immunol. 160, 708-717.

Santiago-Schwarz, F., Belilos, E., Diamond, B., and Carsons, S. E.(1992). TNF in combination with GM-CSF enhances the differentiation ofneonatal cord blood stem cells into dendritic cells and macrophages. J.Leukocyte Biol. 52, 274-281.

Scheicher, C., Mehlig, M., Zecher, R., and Reske, K. (1992). Dendriticcells from mouse bone marrow: in vitro differentiation using low dosesof recombinant granulocyte/macrophage CSF. J. Immunol. Methods 154,253-264.

Schuler, G., Thurner, B., and Romani, N. (1997). Dendritic cells: fromignored cells to major players in T-cell-mediated immunity. Int ArchAllergy Immunol. 112, 317-322.

Seder, R. A., Paul, W. E., Davis, M. M., Fazekas, de St. Groth, B.(1992). The presence of interleukin-4 during in vitro priming determinesthe lymphokine-producing potential of CD4+ T cells from T cell receptortransgenic mice. J. Exp. Med. 176, 1091-1098.

Seder, R. A., Gazzinelli, R., Sher, A., and Paul, W. E. (1993). IL-12acts directly on CD4+ T cells to enhance priming for IFNγ production anddiminishes IL-4 inhibition of such priming. Proc. Natl. Acad. Sci. USA90, 10188-10192.

Serreze, D. V., Leiter, E. H. (1988) Defective activation of Tsuppressor cell function in nonobese diabetic mice. J. Immunol.140:3801-3807.

Serreze, D. V., Gaskins, H. R., Leiter, E. H. (1993) Defects in thedifferentiation and funciton of antigen-presenting cells in NOD/LT mice.J. Immunol. 150:2534-2543.

Steinman, R. M., Pack, M., and Inaba, K. (1997). Dendritic cells in theT-cell areas of lymphoid organs. Imm. Reviews 156, 25-37.

Steinbrink, K., Wolfl, M., Jonuleit, H., Knop, Jurgen, and Enk, A.(1997). Induction of tolerance by IL-10-treated dendritic cells. J.lmmunol. 159, 4772-4780.

Steinman, R. M. 1991. The dendritic cell system and its role inimmunogenicity. Ann. Rev. Immunol. 9:271-96.

Steptoe, R. J., and Thompson, A. W. (1996). Dendritic cells andtolerance induction. Clin. Exp. Immunol. 105, 397-402.

Stewart, T. A., Hultgren, B., Huang, X., Pitts-Meek, S., Hully, J., andMacLachlan, N. J. (1993). Induction of type I diabetes by interferon-ain transgenic mice. Science 260, 1942-1946.

Thompson, C. B. (1995). Distinct roles for the costimulatory ligandsB7-1 and B7-2 in T helper cell differentiation? Cell 81, 979-982.

Trembleau, S., Penna, G., Bosi, E., Mortara, A., Gately, M. K., andAdorini, L. (1995). Interleukin-12 administration induces T helper type1 and accelerates disease in NOD mice. J. Exp. Med. 181, 817-821.

Trinchieri, G. (1995). Interleukin-12: a proinflammatory cytokine withimmunoregulatory functions that bridge innate resistance andantigen-specific adaptive immunity. Annu. Rev. Immunol. 13, 251-276.

Vogel, T., Guo, N. H., Guy, R., Drezlich, N., Krutzsch, H. C., Blake, D.A., Panet, A., and Roberts, D. D. (1994). Apolipoprotein E: a potentinhibitor of endothelial and tumour cell proliferation. J Cell Biochem.54, 299-308.

Wilson, S. B., Kent, S. C., Patton, K. T., Orban, T., Jackson, R. A.,Exley, M., Porcelli, S., Schatz, D. A., Atkinson, M. A., Balk, S. P.,Strominger, J. L., and Hafler, D. A. (1998) Extreme Th1 bias ofinvariant V-alpha-24J-alpha-Q T cells in type 1 diabetes (Letter toNature). Nature 391:177-##.

Wong, B. R., Josien, R., Lee, S. Y., Sauter, B., Li, H.-L., Steinman, R.M., and Choi, Y. (1997). TRANCE (tumor necrosis factor-relatedactivation-induced cytokine), a new TNF family member predominantlyexpressed in T cells, is a dendritic cell-specific survival factor. J.Exp. Med. 186, 2075-2080.

Zipris, D., Crow, A., and Delovitch, T. L. (1991a). Altered thymic andperipheral T lymphocyte repertoire precedes the onset of diabetes in NODmice. Diabetes 40, 429-435.

Zipris, D., Lazarus, A. H., Crow, A., Hadzija, M., and Delovitch, T. L.(1991b). Defective thymic T cells activation by Concavalin A andanti-CD3 in autoimmune nonobese diabetic mice. Evidence for thymic Tcell anergy that correlates with the onset of insulitis. J. Immunol.146, 3763-3771.

20 1 14 PRT Murine 1 Thr Gln Gln Ile Arg Leu Gln Ala Glu Ile Phe Gln AlaArg 1 5 10 2 14 PRT Homo sapiens 2 Ala Gln Gln Ile Arg Leu Gln Ala GluAla Phe Gln Ala Arg 1 5 10 3 14 PRT Artificial Sequence apoEp1.B A240 3Thr Ala Gln Ile Arg Leu Gln Ala Glu Ile Phe Gln Ala Arg 1 5 10 4 14 PRTArtificial Sequence apoEpl.B A241 4 Thr Gln Ala Ile Arg Leu Gln Ala GluIle Phe Gln Ala Arg 1 5 10 5 14 PRT Artificial Sequence apoEpl.B A242 5Thr Gln Gln Ala Arg Leu Gln Ala Glu Ile Phe Gln Ala Arg 1 5 10 6 14 PRTArtificial Sequence apoEpl.B A243 6 Thr Gln Gln Ile Ala Leu Gln Ala GluIle Phe Gln Ala Arg 1 5 10 7 15 PRT Murine 7 Gln Thr Gln Gln Ile Arg LeuGln Ala Glu Ile Phe Gln Ala Arg 1 5 10 15 8 13 PRT Murine 8 Gln Gln IleArg Leu Gln Ala Glu Ile Phe Gln Ala Arg 1 5 10 9 17 PRT Murine 9 Glu GluGln Thr Gln Gln Ile Arg Leu Gln Ala Glu Ile Phe Gln Ala 1 5 10 15 Arg 1016 PRT Murine 10 Glu Gln Thr Gln Gln Ile Arg Leu Gln Ala Glu Ile Phe GlnAla Arg 1 5 10 15 11 12 PRT Murine 11 Gln Ile Arg Leu Gln Ala Glu IlePhe Gln Ala Arg 1 5 10 12 14 PRT Artificial Sequence apoEp1.B A244 12Thr Gln Gln Ile Arg Ala Gln Ala Glu Ile Phe Gln Ala Arg 1 5 10 13 14 PRTArtificial Sequence apoEp1.B A245 13 Thr Gln Gln Ile Arg Leu Ala Ala GluIle Phe Gln Ala Arg 1 5 10 14 14 PRT Artificial Sequence aoEp1.B A247 14Thr Gln Gln Ile Arg Leu Gln Ala Ala Ile Phe Gln Ala Arg 1 5 10 15 14 PRTArtificial Sequence apoEp1.B A248 15 Thr Gln Gln Ile Arg Leu Gln Ala GluAla Phe Gln Ala Arg 1 5 10 16 14 PRT Artificial Sequence apoEp1.B A24916 Thr Gln Gln Ile Arg Leu Gln Ala Glu Ile Ala Gln Ala Arg 1 5 10 17 14PRT Artificial Sequence apoEp1.B A250 17 Thr Gln Gln Ile Arg Leu Gln AlaGlu Ile Phe Ala Ala Arg 1 5 10 18 14 PRT Artificial Sequence apoEp1.BA239, 240 18 Ala Ala Gln Ile Arg Leu Gln Ala Glu Ile Phe Gln Ala Arg 1 510 19 14 PRT Artificial Sequence apoEp1.B A240, 241 19 Thr Ala Ala IleArg Leu Gln Ala Glu Ile Phe Gln Ala Arg 1 5 10 20 14 PRT ArtificialSequence apoEp1.B A239-241 20 Ala Ala Ala Ile Arg Leu Gln Ala Glu IlePhe Gln Ala Arg 1 5 10

We claim:
 1. A method of immune modulation comprising administering aneffective amount of an apoEp1.B peptide or a nucleic acid encoding anapoEp1.B peptide to a cell or animal in need thereof.
 2. A methodaccording to claim 1 wherein the immune modulation is the induction ofcell differentiation.
 3. A method according to claim 2 wherein themethod induces differentiation of a monocyte to a dendritic cell.
 4. Amethod according to claim 2 wherein the cell is a tumor cell.
 5. Amethod according to claim 1 wherein the immune modulation is theinduction of immune tolerance.
 6. A method according to claim 1 whereinthe immune modulation is the inhibition of inflammation.
 7. A methodaccording to claim 6 wherein the immune modulation is the inhibitionatherosclerotic plaque formation.
 8. A method according to claim 1wherein the immune modulation is the treatment of an autoimmune disease.9. A method according to claim 8 wherein the autoimmune disease isdiabetes.
 10. A method according to claim 1 wherein the immunemodulation is the induction of an immune response.
 11. A methodaccording to claim 10 further comprising administering a cytokine to thecell or animal in need thereof.
 12. A method according claim 1 whereinsaid peptide is administered to a cell in vitro.
 13. A method accordingto claim 1 wherein the apoEp1.B peptide (1) has the amino acid sequenceTQQIRLQAEIFQAR (amino acids 239-252) (SEQ.ID.NO.:1); (2) is an analog of(1) wherein the modification occurs in one of amino acids 239-243; (3)is a fragment of (1) or (2) wherein said fragment comprises amino acids240-252; or (4) is an elongation of (1), (2) or (3) wherein theelongation contains an additional amino acid at position
 238. 14. Amethod according to claim 1 wherein the apoEp1.B peptide (1) has theamino acid sequence AQQIRLQAEAFQAR (amino acids 239-252) (SEQ.ID.NO.:2);(2) is an analog of (1) wherein the modification occurs in one of aminoacids 239-243; (3) is a fragment of (1) or (2) wherein said fragmentcomprises amino acids 240-252; or (4) is an elongation of (1), (2) or(3) wherein the elongation contains an additional amino acid at position238.
 15. A method according to claim 1 wherein the apoEp1.B peptide hasthe amino acid sequence TAQIRLQAEIFQAR (SEQ.ID.NO.:3).
 16. A methodaccording to claim 1 wherein the apoEp1.B peptide has the amino acidsequence TQAIRLQAEIFQAR (SEQ.ID.NO.:4).
 17. A method according to claim1 wherein the apoEp1.B peptide has the amino acid sequenceTQQARLQAEIFQAR (SEQ.ID.NO.:5).
 18. A method according to claim 1 whereinthe apoEp1.B peptide has the amino acid sequence TQQIALQAEIFQAR(SEQ.ID.NO.:6).
 19. A method according to claim 1 wherein the apoEp1.Bpeptide has the amino acid sequence QTQQIRLQAEIFQAR (SEQ.ID.NO.:7). 20.A method according to claim 1 wherein the apoEp1.B peptide has the aminoacid sequence QQIRLQAEIFQAR (SEQ.ID.NO.:8).
 21. A method of inducingtolerogenic dendritic cells comprising administering an effective amountof an apoEp1.B peptide or a nucleic acid encoding an apoEp1.B peptide toa cell or animal in need thereof.
 22. A method according to claim 21wherein the apoEp1.B peptide (1) has the amino acid sequenceTQQIRLQAEIFQAR (amino acids 239-252) (SEQ.ID.NO.:1); (2) is an analog of(1) wherein the modification occurs in one of amino acids 239-243; (3)is a fragment of (1) or (2) wherein said fragment comprises amino acids240-252 or (4) is an elongation of (1), (2) or (3) wherein theelongation contains an additional amino acid at position
 238. 23. Amethod according to claim 21 wherein the apoEp1.B peptide (1) has theamino acid sequence AQQIRLQAEAFQAR (amino acids 239-252) (SEQ.ID.NO.:2);(2) is an analog of (1) wherein the modification occurs in one of aminoacids 239-243; (3) is a fragment of (1) or (2) wherein said fragmentcomprises amino acids 240-252 or (4) is an elongation of (1), (2) or (3)wherein the elongation contains an additional amino acid at position238.
 24. A method according to claim 21 wherein the apoEp1.B peptide hasthe amino acid sequence TAQIRLQAEIFQAR (SEQ.ID.NO.:3).
 25. A methodaccording to claim 21 wherein the apoEp1.B peptide has the amino acidsequence TQAIRLQAEIFQAR (SEQ.ID.NO.:4).
 26. A method according to claim21 wherein the apoEp1.B peptide has the amino acid sequenceTQQARLQAEIFQAR (SEQ.ID.NO.:5).
 27. A method according to claim 21wherein the apoEp1.B peptide has the amino acid sequence TQQIALQAEIFQAR(SEQ.ID.NO.:6).
 28. A method according to claim 21 wherein the apoEp1.Bpeptide has the amino acid sequence QTQQIRLQAEIFQAR (SEQ.ID.NO.:7). 29.A method according to claim 21 wherein the apoEp1.B peptide has theamino acid sequence QQIRLQAEIFQAR (SEQ.ID.NO.:8).
 30. A method oftreating a tumor comprising administering an effective amount of anapEp1.B peptide or a nucleic acid encoding an apoEp1.B peptide to ananimal in need thereof.
 31. A method according to claim 30 wherein theapoEp1.B peptide (1) has the amino acid sequence TQQIRLQAEIFQAR (aminoacids 239-252) (SEQ.ID.NO.:1); (2) is an analog of (1) wherein themodification occurs in one of amino acids 239-243; (3) is a fragment of(1) or (2) wherein said fragment comprises amino acids 240-252 or (4) isan elongation of (1), (2) or (3) wherein the elongation contains anadditional amino acid at position
 238. 32. (New) A method according toclaim 30 wherein the apoEp1.B peptide (1) has the amino acid sequenceAQQIRLQAEAFQAR (amino acids 239-252) (SEQ.ID.NO.:2); (2) is an analog of(1) wherein the modification occurs in one of amino acids 239-243; (3)is a fragment of (1) or (2) wherein said fragment comprises amino acids240-252 or (4) is an elongation of (1), (2) or (3) wherein theelongation contains an additional amino acid at position
 238. 33. Amethod according to claim 30 wherein the apoEp1.B peptide has the aminoacid sequence TAQIRLQAEIFQAR (SEQ.ID.NO.:3).
 34. A method according toclaim 13 wherein the apoEp1.B peptide has the amino acid sequenceTQAIRLQAEIFQAR (SEQ.ID.NO.:4).
 35. A method according to claim 30wherein the apoEp1.B peptide has the amino acid sequence TQQARLQAEIFQAR(SEQ.ID.NO.:5).
 36. A method according to claims 13 wherein the apoEp1.Bpeptide has the amino acid sequence TQQIALQAEIFQAR (SEQ.ID.NO.:6).
 37. Amethod according to claim 30 wherein the apoEp1.B peptide has the aminoacid sequence QTQQIRLQAEIFQAR (SEQ.ID.NO.:7).
 38. A method according toclaim 13 wherein the apoEp1.B peptide has the no acid sequenceQQIRLQAEIFQAR (SEQ.ID.NO.:8).
 39. A pharmaceutical composition formodulating an immune response comprising an apoEp1.B peptide or anucleic acid encoding an apoEp1.B peptic in admixture with a suitablediluent or carrier.
 40. A pharmaceutical composition according to claim39 wherein the apoEp1.B peptide (1) has the amino acid sequenceTQQIRLQAEIFQAR (amino acids 239-252) (SEQ.ID.NO.:1); (2) is an analog of(1) wherein the modification occurs in one of amino acids 239-243; (3)is a fragment of (1) or (2) wherein said fragment comprises amino acids240-252; or (4) is an elongation of (1), (2) or (3) wherein theelongation contains an additional amino acid at position
 238. 41. Apharmaceutical composition according to claim 39 wherein the apoEp1.Bpeptide (1) has the amino acid sequence AQQIRLQAEAFQAR (amino acids239-252) (SEQ.ID.NO.:2); (2) is an analog of (1) wherein themodification occurs in one of amino acids 239-243; (3) is a fragment of(1) or (2) wherein said fragment comprises amino acids 240-252; or (4)is an elongation of (1), (2) or (3) wherein the elongation contains anadditional amino acid at position
 238. 42. A pharmaceutical compositionaccording to claim 39 wherein the apoEp1.B peptide has the amino acidsequence TAQIRLQAEIFQAR (SEQ.ID.NO.:3).
 43. A pharmaceutical compositionaccording to claim 39 wherein the apoEp1.B peptide has the amino acidsequence TQAIRLQAEIFQAR (SEQ.ID.NO.:4).
 44. A pharmaceutical compositionaccording to claim 39 wherein the apoEp1.B peptide has the amino acidsequence TQQARLQAEIFQAR (SEQ.ID.NO.:5).
 45. A pharmaceutical compositionaccording to claim 39 wherein the apoEp1.B peptide has the amino acidsequence TQQIALQAEIFQAR (SEQ.ID.NO.:6).
 46. A pharmaceutical compositionaccording to claim 39 wherein the apoEp1.B peptide has the amino acidsequence QTQQIRLQAEIFQAR (SEQ.ID.NO.:7).
 47. A pharmaceuticalcomposition according to claim 39 wherein the apoEp1.B peptide has theamino acid sequence QQIRLQAEIFQAR (SEQ.ID.NO.:8).
 48. A pharmaceuticalcomposition according to claim 39 further comprising a cytokine.
 49. Anisolated apoEp1.B peptide (1) having the amino acid sequenceAQQIRLQAEAFQAR (amino acids 239-252) (SEQ.ID.NO.:2); (2) is an analog of(1) wherein the modification occurs in one of amino acids 239-243; (3)is a fragment of (1) or (2) wherein said fragment comprises amino acids240-252; or (4) is an elongation of (1), (2) or (3) wherein theelongation contains an additional amino acid at position
 238. 50. Anisolated apoEp1.B peptide having the amino acid sequence TAQIRLQAEIFQAR(SEQ.ID.NO.:3).
 51. An isolated apoEp1.B peptide having the amino acidsequence TQAIRLQAEIFQAR (SEQ.ID.NO.:4).
 52. An isolated apoEp1.B peptidehaving the amino acid sequence TQQARLQAEIFQAR (SEQ.ID.NO.:5).
 53. Anisolated apoEp1.B peptide having the amino acid sequence TQQIALQAEIFQAR(SEQ.ID.NO.:6).
 54. An isolated apoEp1.B peptide having the amino acidsequence QTQQIRLQAEIFQAR (SEQ.ID.NO.:7).
 55. An isolated apoEp1.Bpeptide having the amino acid sequence QQIRLQAEIFQAR (SEQ.ID.NO.:8).